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Total antioxidant status of human colostrum, transitory milk and mature milk in healthy postpartum women from coastal Croatia
S102
Abstracts / Toxicology Letters 238S (2015) S56–S383
http://dx.doi.org/10.1016/j.toxlet.2015.08.335
P03-046 Recommendation of the reconstructed 3-D skin micronucleus assay (RSMN assay) as an in vitro alternative confirmatory micronucleus assay D. Kidd ∗ , T. Chirom, H. Lyon, S. Phillips, R. Smith, D. Wood, J. Whitwell, J. Clements Covance Laboratories Ltd, Genetic Toxicology, Harrogate, United Kingdom The testing and marketing bans within the EU Directive for Cosmetics has driven much research into suitable in vitro alternative assays to support satisfactory risk assessments. One such assay is the reconstructed skin micronucleus (RSMN) assay. First reported in 2006 (Curren et al.), further development occurred and a standard protocol was published (Dahl et al., 2011). To validate the assay at Covance, eight chemicals (incl. three negatives, two requiring metabolic activation, one aneugen) were selected from the available literature and tested using the 48 h treatment regimen in the presence of cytochalasin B (compound addition: 0 h and 24 h) with mitomycin C as positive control. 48 h post treatment initiation, cells were harvested from the 3-D tissues, fixed, slides prepared, scored for replication index, then coded and scored for micronuclei. Mitomycin C, Methyl methanesulfonate, Vinblastine and Ethyl nitrosourea all yielded statistically significant increases in micronuclei. 4-nitrophenol and 2-ethyl-1,3-hexanediol were negative (as expected). Consistent with published data, Cyclohexanone yielded a non-linear response and Benzo[a]pyrene was negative after 48 h (Aardema et al., 2011). Also, Cyclophosphamide yielded a single positive data point (non-linear trend, also observed by Aardema et al., 2011) using the extended 72 h treatment regimen (treatment at 0, 24 and 48 h). We have shown that this assay system detects direct acting mutagens (including aneugens) but from the limited data set (and consistent with skin’s limited metabolic activity), the ability to detect metabolically activated compounds is questionable. Work is continuing in-house (and elsewhere) to further investigate and optimise the test system (e.g. using longer treatment periods). Given the concordance of these data with the published literature, and in conjunction with our additional recommendations, the assay is considered suitable for use in our laboratory. http://dx.doi.org/10.1016/j.toxlet.2015.08.336
P03-047 Total antioxidant status of human colostrum, transitory milk and mature milk in healthy postpartum women from coastal Croatia M. Matek Saric 1,∗ , J. Grzunov Letinic 2 , J. Jurasovic 3 , T. Orct 3 , A. Sulimanec 3 , V.M. Varnai 3 , M. Piasek 3 1
University of Zadar, Department of Health Studies, Zadar, Croatia Institute of Public Health Zadar, Zadar, Croatia 3 Inst. Med. Res. & Occup. Health, Zagreb, Croatia 2
Purpose: Health risk of exposure to three major toxic metals Cd, Pb and Hg were assessed in healthy breastfeeding women aged 29.5 ± 4.9 (19–44 yrs) who gave birth during 2012/13 in the maternity ward of General County Hospital in Zadar, the largest city in mid-coastal Croatia. Data on smoking and dietary habits (with focus
on seafood consumption) by self-ascertainment and stated number of amalgam dental fillings as sources of toxic metal exposure were collected by questionnaire. Metal levels in maternal blood and breast milk samples (elements were analysed by ICP–MS) served as biomarkers of exposure. As biomarkers of toxic metal effects, we measured total antioxidant status (TAS) in breast milk samples (in total N = 147) of three lactation stages: colostrum (up to day 4), transitory milk (between days 5 and 14) and mature milk (between days 20 and 30 postpartum) by Antioxidant Assay Kit (Cayman Chemical Company, USA) based upon the ability of antioxidants to inhibit the oxidation of ABTS. Results: Maternal cigarette smoking was positively correlated with Cd concentrations in maternal blood and mature milk and Pb in transitory milk. Positive correlations were also found between seafood consumption and total Hg in maternal blood and mature milk and between the number of maternal amalgam dental fillings and total Hg in colostrum and transitory milk. Values of TAS (expressed in mmol/L of Trolox equivalents) determined in colostrum, transitory milk and mature milk samples were: 1.35 ± 0.74, 1.49 ± 0.79 and 1.48 ± 0.80. Conclusions: The absence of differences in TAS values between different breast milk types in relation to maternal smoking and dietary habits suggest that exposure to potential oxidative agents, including three main toxic metals, in the study cohort did not reach levels to disrupt the protective antioxidant capacity of breast milk, which might point to possible health risks in postpartum women and their progeny. http://dx.doi.org/10.1016/j.toxlet.2015.08.337
P03-048 Human health risk assessment of boric acid and sodium borates Y. Duydu 1,∗ , N. Basaran 2 , B.M. Hermann 3 1
Ankara University, Faculty of Pharmacy, Toxicology, Ankara, Turkey Hacettepe University, Faculty of Pharmacy, Toxicology, Ankara, Turkey 3 Leipzig Research Centre for Working Environment and Human Factors, Dortmund, Germany 2
Boric acid and sodium borates are classified as toxic to reproduction in the CLP regulation under “Category 1B” with the hazard statement of “H360FD” due to the reprotoxic effects in animal studies at high doses. However, the results of the environmental and occupational epidemiology studies published so far do not support these findings in humans. Our study was performed in boric acid production plant located in Bandırma, Turkey with the aim of investigating the boron mediated reprotoxic effects in highly boron exposed mail workers (n = 204). However, boron mediated unfavorable effects on reproduction in male workers have not been reported even in the workers under very high daily boron exposure conditions. The results of epidemiological studies should be analyzed more in depth to understand the underlying reason of this inconsistency between animal experiments and epidemiological study results. The NOAEL for the reprotoxic effects of boron in rats is 17.5 mg/kg-bw/day. Accordingly the RfD of boron is 0.29 mg/kg-bw/day when using “60 (17.5/60)” as the chemical specific composite uncertainty factor for boric acid. The mean daily boron intake/exposure level of the high exposure group (0.21 mg B/kg-bw/day) in boric acid production plant (Turkey) was lower than the RfD computed for boron. The NOAEL for rat reproductive toxicity is equivalent to a blood boron level of 2020 ng/g. This level is higher than the mean blood boron concentration (223.89 ± 69.49 ng/g) of the high exposure group workers in boric acid production plant (Turkey) by a factor of 9. From this point of
Abstracts / Toxicology Letters 238S (2015) S56–S383
http://dx.doi.org/10.1016/j.toxlet.2015.08.335
P03-046 Recommendation of the reconstructed 3-D skin micronucleus assay (RSMN assay) as an in vitro alternative confirmatory micronucleus assay D. Kidd ∗ , T. Chirom, H. Lyon, S. Phillips, R. Smith, D. Wood, J. Whitwell, J. Clements Covance Laboratories Ltd, Genetic Toxicology, Harrogate, United Kingdom The testing and marketing bans within the EU Directive for Cosmetics has driven much research into suitable in vitro alternative assays to support satisfactory risk assessments. One such assay is the reconstructed skin micronucleus (RSMN) assay. First reported in 2006 (Curren et al.), further development occurred and a standard protocol was published (Dahl et al., 2011). To validate the assay at Covance, eight chemicals (incl. three negatives, two requiring metabolic activation, one aneugen) were selected from the available literature and tested using the 48 h treatment regimen in the presence of cytochalasin B (compound addition: 0 h and 24 h) with mitomycin C as positive control. 48 h post treatment initiation, cells were harvested from the 3-D tissues, fixed, slides prepared, scored for replication index, then coded and scored for micronuclei. Mitomycin C, Methyl methanesulfonate, Vinblastine and Ethyl nitrosourea all yielded statistically significant increases in micronuclei. 4-nitrophenol and 2-ethyl-1,3-hexanediol were negative (as expected). Consistent with published data, Cyclohexanone yielded a non-linear response and Benzo[a]pyrene was negative after 48 h (Aardema et al., 2011). Also, Cyclophosphamide yielded a single positive data point (non-linear trend, also observed by Aardema et al., 2011) using the extended 72 h treatment regimen (treatment at 0, 24 and 48 h). We have shown that this assay system detects direct acting mutagens (including aneugens) but from the limited data set (and consistent with skin’s limited metabolic activity), the ability to detect metabolically activated compounds is questionable. Work is continuing in-house (and elsewhere) to further investigate and optimise the test system (e.g. using longer treatment periods). Given the concordance of these data with the published literature, and in conjunction with our additional recommendations, the assay is considered suitable for use in our laboratory. http://dx.doi.org/10.1016/j.toxlet.2015.08.336
P03-047 Total antioxidant status of human colostrum, transitory milk and mature milk in healthy postpartum women from coastal Croatia M. Matek Saric 1,∗ , J. Grzunov Letinic 2 , J. Jurasovic 3 , T. Orct 3 , A. Sulimanec 3 , V.M. Varnai 3 , M. Piasek 3 1
University of Zadar, Department of Health Studies, Zadar, Croatia Institute of Public Health Zadar, Zadar, Croatia 3 Inst. Med. Res. & Occup. Health, Zagreb, Croatia 2
Purpose: Health risk of exposure to three major toxic metals Cd, Pb and Hg were assessed in healthy breastfeeding women aged 29.5 ± 4.9 (19–44 yrs) who gave birth during 2012/13 in the maternity ward of General County Hospital in Zadar, the largest city in mid-coastal Croatia. Data on smoking and dietary habits (with focus
on seafood consumption) by self-ascertainment and stated number of amalgam dental fillings as sources of toxic metal exposure were collected by questionnaire. Metal levels in maternal blood and breast milk samples (elements were analysed by ICP–MS) served as biomarkers of exposure. As biomarkers of toxic metal effects, we measured total antioxidant status (TAS) in breast milk samples (in total N = 147) of three lactation stages: colostrum (up to day 4), transitory milk (between days 5 and 14) and mature milk (between days 20 and 30 postpartum) by Antioxidant Assay Kit (Cayman Chemical Company, USA) based upon the ability of antioxidants to inhibit the oxidation of ABTS. Results: Maternal cigarette smoking was positively correlated with Cd concentrations in maternal blood and mature milk and Pb in transitory milk. Positive correlations were also found between seafood consumption and total Hg in maternal blood and mature milk and between the number of maternal amalgam dental fillings and total Hg in colostrum and transitory milk. Values of TAS (expressed in mmol/L of Trolox equivalents) determined in colostrum, transitory milk and mature milk samples were: 1.35 ± 0.74, 1.49 ± 0.79 and 1.48 ± 0.80. Conclusions: The absence of differences in TAS values between different breast milk types in relation to maternal smoking and dietary habits suggest that exposure to potential oxidative agents, including three main toxic metals, in the study cohort did not reach levels to disrupt the protective antioxidant capacity of breast milk, which might point to possible health risks in postpartum women and their progeny. http://dx.doi.org/10.1016/j.toxlet.2015.08.337
P03-048 Human health risk assessment of boric acid and sodium borates Y. Duydu 1,∗ , N. Basaran 2 , B.M. Hermann 3 1
Ankara University, Faculty of Pharmacy, Toxicology, Ankara, Turkey Hacettepe University, Faculty of Pharmacy, Toxicology, Ankara, Turkey 3 Leipzig Research Centre for Working Environment and Human Factors, Dortmund, Germany 2
Boric acid and sodium borates are classified as toxic to reproduction in the CLP regulation under “Category 1B” with the hazard statement of “H360FD” due to the reprotoxic effects in animal studies at high doses. However, the results of the environmental and occupational epidemiology studies published so far do not support these findings in humans. Our study was performed in boric acid production plant located in Bandırma, Turkey with the aim of investigating the boron mediated reprotoxic effects in highly boron exposed mail workers (n = 204). However, boron mediated unfavorable effects on reproduction in male workers have not been reported even in the workers under very high daily boron exposure conditions. The results of epidemiological studies should be analyzed more in depth to understand the underlying reason of this inconsistency between animal experiments and epidemiological study results. The NOAEL for the reprotoxic effects of boron in rats is 17.5 mg/kg-bw/day. Accordingly the RfD of boron is 0.29 mg/kg-bw/day when using “60 (17.5/60)” as the chemical specific composite uncertainty factor for boric acid. The mean daily boron intake/exposure level of the high exposure group (0.21 mg B/kg-bw/day) in boric acid production plant (Turkey) was lower than the RfD computed for boron. The NOAEL for rat reproductive toxicity is equivalent to a blood boron level of 2020 ng/g. This level is higher than the mean blood boron concentration (223.89 ± 69.49 ng/g) of the high exposure group workers in boric acid production plant (Turkey) by a factor of 9. From this point of